The overall goal of this proposal is to investigate the role of adenosine (ADO) in cerebral blood flow (CBF) regulation. ADO is a purine nucleoside and breakdown product of ATP via AMP. In our most recent grant, we (1) defined the ADO receptor subtype involved in vasodilation induced by ADO in vitro and during cortical activation evoked by sciatic nerve stimulation (SNS) in vivo; (2) determined that diffusion and counter current mechanisms are not involved in pial vasodilation during SNS; (3) discovered that glutamate-induced vasodilation is ADO receptor-mediated; (4) found that ADO-induced vasodilation probably involves a cGMP dependent protein kinase (PKG) and cGMP, and not cAMP-dependent protein kinase (PKA) and cAMP; (5) determined that ADO receptor manipulations during ischemia affect both brain injury and learning/memory and (6) found a novel neuroprotective effect of ADO that decreases excitatory amino acid (EAA) uptake into the brain. The present proposal will focus on the cellular and molecular mechanisms involved in ADO regulation of CBF. We will use a comprehensive multidisciplinary approach involving genetically engineered mice, in vivo and in vitro vessel preparations, cell culture, electrophysiologic, biochemical and molecular techniques. Specific Aims: To test the following hypotheses (1) that A2B receptor activation contributes to ADO-induced and SNSinduced vasodilation, (2) that endothelial cells or vascular smooth muscle mediate ADO-induced conduction of dilator signals along cerebral arterioles, (3) that with glutamate stimulation, astrocytes are a cellular source for ADO release, (4) that in the cerebral circulation, ADO-induced vasodilation is mediated by PKG, (5) that ADO, via a receptor subtype mechanism, decreases excitatatory amino acid transport into the brain. Further investigation of ADO in brain will allow more comprehensive knowledge of the regulation of cerebral blood flow and the link between metabolism and vascular response. Such knowledge is necessary to allow more rational and innovative treatments of brain ischemia, head injury and stroke.